The present invention relates to methods of delivering a nucleic acid to motor neurons comprising administering the nucleic acid to muscle tissue. More particularly, the invention relates to methods for treating pathologies of the nervous system by intramuscular administration of a therapeutic gene, and gene transfer into medullary motor neurons. This invention also relates to compositions comprising the gene in a form suitable for intramuscular administration.
Neurodegenerative Disease
Motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy of infancy (SMA), are often debilitating and resist curative treatment. For example, ALS is heavily disabling and invariably lethal. With an incidence of 2.5/100 000 in constant increase, a prevalence of 6-10/100,000 (Leigh, Pathogenic mechanisms in amyotrophic lateral sclerosis and other motor neurons disorders in Neurodegenerative diseases by CALNE, Saunders W. B. Eds, Philadelphia, U.S.A., 1994), ALS affects 90,000 patients in developed countries, mostly adults in their sixth decade. The disease is characterized by a progressive motor neuron degeneration leading to paralysis, to total loss of motor and respiratory functions, and eventually to death two to eight years after the appearance of the first clinical signs (mean duration after onset three years). ALS is of genetic origin in 5% of the patients, and sporadic in 95% of the cases. Point mutations in the gene encoding for Cu/Zn superoxide dismutase (SOD1) localised on chromosome 21q22-1 are responsible for the pathology in 20% of the familial cases (Rosen et al., Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature, 362, 59-62, 1993, review in Rowland, Amyotrophic lateral sclerosis: Human challenge for neuroscience, Proc. Natl. Acad. Sci. USA, 92, 1251-1253, 1995). The pathophysiological basis of the sporadic forms remains unknown. Although the use of Rilutek provides ALS patients with a modest increase in survival probability, there is no curative treatment available for this disorder.
Spinal muscular atrophy of infancy is an autosomal recessive disease, which in its most severe form (SMA type 1) affects 1/16,000-25 000 infants in Europe and North America. SMA-1 patients manifest weakness before three months of age and are never able to be maintained in a sitting posture. Average life expectancy is 8 months, with 95% mortality before the second birthday (review in Crawford and Pardo, The neurobiology of childhood spinal muscular atrophy, Neurobiol. of Disease, 3, 97-110, 1996). The disease is linked to mutations in the SMN gene (Lefebvre et al., Identification and characterization of a spinal muscular atrophy determining gene, Cell, 80, 155-165, 1995). There is no curative treatment available for this disease.
Neurothrophic Factors
Neurotrophic factors have been suggested as potential therapeutic agents for motor neuron diseases (Thoenen et al., Exp. Neurology 124,47-55, 1993). Indeed, embryonic motor neuron survival in culture is enhanced by members of the neurotrophin family such as brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4 (NT-4/5), cytokines such as ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF) and cardiotrophin-1, glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor-1 (IGF-1) and members of the FGF family (review in Henderson, Neurotrophic factors as therapeutic agents in amyotrophic lateral sclerosis: potential and pitfalls. In Serratrice G. T. and Munsat T. L. eds. Pathogenesis and therapy of amyotrophic lateral sclerosis. Advances in Neurology, 68, pp. 235-240, 1995. Lippincott-Raven publishers, Philadelphia; Pennica et al., Cardiotrophin-l a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons. Neuron, 17, 63-74, 1996).
In vivo, a reduction of motoneuronal death occurring naturally during embryonic development was observed with CNTF (Oppenheim et al., Control of embryonnic motoneuron survival in vivo by ciliary neurotrophic factor. Science, 251, 1616-1618, 1991), BDNF (Oppenheim et al., Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death. Nature, 360, 755-757, 1992), GDNF (Oppenheim et al., Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature, 373, 344-346, 1995), and cardiotrophin-1 (Pennica et al., 1996). Protection from retrograde motor neuron death after acute peripheral nerve axotomy in neonate rodents was evidenced with several factors (Sendtner et al., Ciliary neurotroptuc factor prevents the degeneration of motor neurons after axotomy, Nature 345, 440-441, 1990, Sendtner et al., Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy. Nature, 358, 502-504, 1992; Sendtner et al., Brain-derived neurotrophic prevents the death of motoneurons in newborn rats after nerve section. Nature, 360, 757-759, 1992; Vejsada et al., Quantitative comparison of the transient rescue effects of neurotrophic factors on axotomised motoneurons in vivo. Eur. J. Neurosci., 7, 108-115, 1995). Also, a protective effect of CNTF and/or BDNF was described in two murine models of inherited progressive motor degeneration (Sendtner et al., 1992; Mitsumoto et al., Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF. Science, 265, 1107-1110, 1994).
Data showing that neurotrophic factors enhance motoneuronal survival under multiple experimental conditions, suggest that these molecules could decrease the vulnerability of motoneurons in human pathologies. However the use of trophic factors in patients is limited by their poor bioavailaility after systemic administration.
Neurotrophic factors systemically administered penetrate the nervous system with low yield because of the presence of the blood-brain barrier. Only a very small fraction of the injected factor reaches motoneurons, most probably at the level of neuromuscular synapses (Dittrich et al., Ciliary neurotrophic cancer: pharacokinetics and acute phase response. Ann. Neurol., 35, 151-163, 1994, Yan et al., 1994). Furthermore trophic factors are rapidly degraded and display a short half-life after systemic administration (2.9 minutes for CNTF in rat Dittrich et al., 1994, Cedarbaum et al., A double-blind placebo-controlled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in amyotrophic lateral sclerosis. Neurology, 46, 1244-1249, 1996). As a consequence high doses have to be administered in order to have any possibility of reaching therapeutic concentrations at the motoneuronal levels. However, such doses are likely to produce negative side effects. This has been illustrated in clinical trials using recombinant CNTF injected to ALS patients. The injected doses shown to produce a therapeutic effect in animals (Mitsumoto et al, 1994) were above toxicity threshold in human (Miller et al., A placebo-controlled trial of recombinant human ciliary neurotrophic (rhCNTF) factor in amyotrophic lateral sclerosis. Annals Neurol., 39, 256-260, 1996), and adverse effects such as cough, asthenia, nausea, weight loss and even increased death rate at the highest dose were observed, while no beneficial effect of CNTF treatment could be detected (Miller et al., 1996).
The clinical use of neurotrophic factors thus requires the development of suitable modes of in vivo delivery. Therapeutic gene transfer offers potential advantages over direct administration of the protein, such as continuous and/or targeted production of the desired transgene in vivo.
Gene Therapy
Gene therapy is rapidly emerging as an effective approach for management and treatment of a variety of diseases. Examples of effective gene therapy regimens appear routinely in the literature (see for example Roth et al., Nature Medicine, Vol. 2, 985-991 (1996)).
Gene therapy, including the administration of modified viruses as vectors, constitutes a particularly promising approach for treating neurodegenerative diseases. Among the viruses in current use for gene therapy are adenoviruses (Le Gal La Salle et al., Science 259, 988-990), herpes viruses, adeno-associated viruses and retroviruses. Studies have shown that these vectors, and in particular the adenoviruses, are capable of infecting with a very high efficiency cells of the central nervous system. These results have enabled the development of methods for treating pathologies of the central nervous system by direct injection into the central nervous system (in particular by stereotaxis) of recombinant adenoviruses comprising a therapeutic gene (see WO94/08026, the contents of which are incorporated herein by reference).
With respect to neurodegenerative diseases or traumas associated with the spinal cord, gene therapy provides a method to combat degeneration of the motor neurons (motoneurons) by delivering therapeutic genes, such as a gene encoding a neurotrophic factor or a growth factor to motor neurons. However, prior methods are limited by the lack of a simple method enabling specific transfer of a gene into motor neurons. The present invention overcomes this problem.
The present invention describes a particularly efficient method for the selective transfer of genes into motor neurons.
One aspect of the invention provides a method of delivering a nucleic acid to mammalian motor neurons comprising administering said nucleic acid to muscle tissue, wherein said nucleic acid is delivered to said motor neurons.
Another aspect of the invention is a method of delivering a nucleic acid to mammalian motor neurons comprising administering said nucleic acid to muscle tissue proximate to the site of a nerve linkage associated with a chosen medullary functional level, wherein said nucleic acid is delivered to said motor neurons.
Another aspect of the invention is a method of producing a protein in mammalian motor neurons comprising administering a nucleic acid encoding said protein to muscle tissue, wherein said nucleic acid is delivered to said motor neurons and expressed. In a preferred embodiment, the protein is produced at the post synaptic ends of neuromuscular junctions.
The invention also provides a method of inducing peripheral or collateral sprouting of motor axon endings comprising administering a nucleic acid to muscle tissue, wherein said nucleic acid induces peripheral or collateral sprouting of motor axon endings. In a preferred embodiment, the nucleic acid encodes a protein, such as a neurotrophic factor or a growth factor.
Another aspect of the invention is a method of protecting against axonal degeneration comprising administering a nucleic acid to mammalian muscle tissue, wherein said nucleic acid is delivered to said motor neurons and protects against axonal degeneration.
Still another aspect of the invention is a method of treating an impairment of the nervous system comprising administering a nucleic acid encoding a neuroactive substance to mammalian muscle tissue, wherein said nucleic acid is delivered to motor neurons. Preferred embodiments are the treatment of nerve damage and neurodegenerative diseases, such as amyotrophic lateral sclerosis and spinal muscular atrophy of infancy.
A preferred aspect of the invention is a method of treating amyotrophic lateral sclerosis comprising administering to muscle tissue of a mammal suffering therefrom a replication defective adenovirus comprising a gene encoding neurotrophin-3.